Altered assembly, function, and plasticity of synapses and neural circuits underlie cognitive, memory, and emotional deficits of essentially all neuropsychiatric and neurological diseases. A well-investigated mechanism that regulates synaptic protein turnover and synapse remodeling is the conventional ubiquitin-proteasome pathway, by which polyubiquitin chains conjugate to protein substrates (likely through lysine 48 (K48) of ubiquitin) and tag them for proteasomal degradation. A vastly overlooked ubiquitin modification in the central nervous system (CNS) is K63-linked polyubiquitination, an unconventional linkage mechanism generally believed not to target proteasomal degradation; rather, it regulates protein scaffolding, trafficking, and activity. Despite its recognized importace in cellular signaling mechanisms that mediate innate and adaptive immunity, virtually nothing is known about the role of this proteasome-independent polyubiquitination process in the CNS, especially synapses. Nonetheless, K63-linked ubiquitination is the second most abundant linkage in the rat brain, only slightly behind the K48 linkage. In addition, emerging evidence points to a link between K63-linked ubiquitination and a number of brain disorders. Thus, there is a need to investigate the mechanisms and roles of this nonproteolytic polyubiquitin topology in neurons and at synapse. Our preliminary studies indicate that K63-linked polyubiquitination is a fundamental mechanism regulating synapse assembly and plasticity and identify the postsynaptic scaffolding protein PSD-95 as the first substrate. We also identify a PSD-associated, K63-linkage-specific enzyme machinery that controls PSD-95 ubiquitination at synapses. The goals of this R01 application are to define the molecular details and functional consequences of PSD-95 K63 polyubiquitination (Aim 1), to delineate the role of K63 polyubiquitination in synapse development, function, remodeling, and plasticity (Aim 2), and to characterize the behavioral alterations in mice lacking K63-linkage-specific E3 ubiquitin ligase TRAF6 and deubiquitinase CYLD (Aim 3). A combination of molecular, biochemical, electrophysiological, and behavioral approaches will be employed. The proposed studies represent a fundamentally important conceptual breakthrough that opens up new avenues for investigation of synapse and circuit function and plasticity. The project has the potential to uncover new paradigm-shifting principles that govern neuro-immune interactions, which may contribute to abnormal brain wiring in neurodevelopmental disorders (such as schizophrenia and autism spectrum disorders) and neural circuit repair in the adult brain following injury. The information obtained will facilitate development of novel treatment strategies for these brain disorders.